Peptide nucleic acid (PNA) is a DNA mimic that recognizes complementary sequences by Watson-Crick base-pairing. One of the strengths of PNAs is their ability to recognize sites within duplex DNA by strand invasion. We have developed efficient methods for synthesizing PNAs and introducing them into cells. We have also characterized strategies for improving the efficiency of strand invasion. We now propose to combine these advances to develop antigene PNAs as agents for manipulating gene expression at the level of the chromosome. The objective of this proposal is to understand the properties of antigene PNAs and test the hypothesis that antigene PNAs can recognize chromosomal DNA inside cells. Specifically, we propose to 1) Characterize and optimize the intracellular localization of PNAs, 2) Develop rules for using antisense PNAs to manipulate gene expression, and 3) Explore the value of antigene PNAs as a general strategy for recognizing duplex DNA by targeting genes that are important in the progression of cancer, specifically c-myc and the reverse transcriptase component of telomerase h TER T. To achieve these goals my laboratory will take advantage of our ability to rapidly synthesize PNAs and PNA-peptide conjugates and our experience with using PNAs inside cells. Preliminary experiments have already shown that PNAs can act as antigene agents, providing a powerful starting point for the experiments described in this proposal. Compounds that sequence-specifically recognize chromosomal DNA have enormous potential to treat disease and be powerful research tools. Such agents might block gene expression, activate gene expression, or enable harmful mutations to be corrected. In spite of the attractiveness of chromosomal DNA as a target, the development of antigene agents has been slow. The studies described in this proposal rigorously test the value of antigene PNAs for the recognition of chromosomes and our data will shape decisions regarding the use of PNAs for laboratory studies and clinical development.